A. Field of the Invention
The present invention relates to the field of cancer therapy, specifically, the treatment of Bcl-2 diseases. More particularly, these treatments involve the use of antisense oligodeoxynucleotides and liposomal formulations thereof.
B. Related Art
Bcl-2 has been linked to a wide variety of diseases such as hematologic malignancies, both leukemias and lymphomas, including follicular and nonfollicular lymphomas, chronic lymphocytic leukemia, and plasma cell dyscrasias (Campos et al., Blood, 84:595, 1994); solid tumors like those associated with breast, prostate and colon cancer; and immune disorders. One particular Bcl-2-related disease is Follicular non-Hodgkin Lymphoma (FL). FL is the most common lymphoid malignancy in Europe and the United States. Typically it is an indolent, low grade disease consisting of an accumulation of small, resting B cells. Although the response to chemotherapy is initially good, relapses are inevitable with the transformation to a more aggressive histological type and the development of drug resistance (Aisenberg, J. Clin. Oncol., 13:2656, 1995; Johnson et. al, J. Clin. Oncol., 13:140, 1995). In over 90% of FL patients, a t(14;18) translocation is found, which results in the juxtaposition of the bcl-2 gene from chromosome 18q21 with the immunoglobulin heavy chain gene locus on chromosome 14q323 (Tsujimoto et. al, Science, 229:1390, 1985; Graninger et. al, J. Clin. Invest., 80:1512, 1987). As a consequence, the bcl-2 gene is under the influence of immunoglobulin heavy chain enhancer, and the Bcl-2 protein is overexpressed (Bakhshi et. al, Cell, 41:899, 1985; Tsujimoto et. al, Oncogene, 2:3, 1987). Bcl-2 tumorigenic potential is related to its capacity of interfering with physiological death responses, thereby enhancing the longevity of the cell (Nuñez et. al, J. Immunol., 144:3602, 1990). The Bcl-2 protein blocks apoptotic stimuli such as growth factor deprivation, radiation, heat-shock, virus, and most of the chemotherapeutic agents (Reed, Hematol. Oncol. Clin. North Am., 9:451, 1995; Hockenbery et. al, Nature, 348:334, 1990). In bcl-2-Ig-transgenic mice, a polyclonal follicular lymphoproliferation consisting of an expansion of mature B lymphocytes is initially observed (McDonnell et. al, Cell, 57:79, 1989). Subsequently, a monoclonal high grade large immunoblastic type lymphomas develop with 50% of them presenting rearrangement of C-MYC. This suggests that a second genetic alteration is necessary for the development and progression of malignant lymphoma (McDonnell and Korsmeyer, Nature, 349:254, 1991).
Recently, an expanding family of Bcl-2-related proteins has been identified. This includes Bax, Bcl-XL, Bcl-XS, Bad, Bak, Mcl-1, A-1, and several open reading frames in DNA viruses (Oltvai et. al, Cell, 74:609, 1993; Boise et. al, Cell, 74:597, 1993; Yang et. al, Cell, 80:285, 1995; Chittenden et. al, Nature, 374:733, 1995; Kiefer et. al, Nature, 374: 736, 1995; Kozopas et. al Proc. Nat'l Acad. Sci. USA, 90:3516, 1993; Lin et. al, J. Immunol., 151:1979, 1993; Pearson et. al, Virology, 160:151, 1987; Neilan et. al, J. Virol., 67:4391, 1993). Membership in the Bcl-2 family of proteins is principally defined by homology within the BH1 and BH2 domains, which help regulate dimerization between the members (Sato et. al, Proc. Nat'l Acad. Sci. USA, 91:9238, 1994). Bax, which shares 21% amino-acid identity with Bcl-2, can bind to Bcl-2 protein and neutralize its ability to block cell death. Thus, the ratio of Bcl-2 to Bax is thought to determine the cell's susceptibility to death following an apoptotic stimulus (Oltvai et. al, 1993; Yin et. al, Nature, 369: 321, 1994).
Phosphodiester antisense oligodeoxynucleotides complementary to specific sequences of the translation-initiation site of Bcl-2 mRNA are able to inhibit the production of the Bcl-2 protein and the growth of t(14;18) translocation bearing cells (Kitada et. al, Antisense Res. Dev., 3:157, 1993). However, the therapeutic use of antisense oligonucleotides has been hampered by their low cellular uptake and their rapid degradation by nucleases and other serum or cellular components. Phosphorothioate oligonucleotides, which are resistant to nuclease degradation, were found to inhibit FL cell growth at concentrations 10 times lower than phosphodiester oligonucleotides (Reed et. al, Cancer Research, 50: 6565, 1990a; Cotter et. al, Oncogene, 9:3049, 1994). However, this approach suffers from low cellular uptake of the oligonucleotides. For example, Reed et. al had to use concentrations of greater than 25 μM of phosphorothioates to achieve 50% growth inhibitions of cell lines derived from B-cell lymphomas, such as 697 and Su-Dhl-4 cells. Liposomal incorporation has led to enhanced uptake of oligonucleotides into leukemic cells (Akhtar et. al, Nucleic Acids Res., 19:5551, 1991; Tari et. al, Blood, 84:601, 1994). The use of cationic lipids by Reed et. al to deliver phosphorothioate antisense oligonucleotides allowed them to reduce the concentration of oligonucleotides to 0.075 to 0.3 μM and still induce growth inhibition in Su-Dhl-4 cells.
There is still, however, a great need for methods and compositions for the treatment of Bcl-2 associated diseases such as hematologic malignancies, both leukemias and lymphomas, including follicular and nonfollicular lymphomas, chronic lymphocytic leukemia, and plasma cell dyscrasias; solid tumors like those associated with breast, prostate and colon cancer; and immune disorders.